The inducible nitric oxide synthase (iNOS) is upregulated diffusely during sepsis and clearly contributes to hemodynamic instability and organ injury during septic shock. However, in stark contrast to the damaging effects of iNOS in many tissues, in the liver upregulation of iNOS has a protective function. Sustained, high-level iNOS expression has no toxic effects on hepatocytes, and even low-level expression is protective. We now hypothesize that upregulation of iNOS in the liver during acute inflammatory states (i.e., sepsis) is part of a protective response that limits the toxicity of pro-inflammatory mediators, including TNFalpha. We have shown that NO can directly inhibit TNFalpha-induced signaling pathways leading to hepatocyte death. By activating soluble guanylate cyclase, NO inhibits the activation of the caspase cascade by TNFalpha, and via S-nitrosylation NO directly inhibits caspase protease activity. We have also shown that NO can regulate gene expression in hepatocytes to promote cell survival. We will now pursue the mechanistic basis of these observations in three aims. AIM I: To determine how NO/cGMP/G-kinase inhibits TNFalpha signaling in hepatocytes. Experiments under Aim I will define the level at which cGMP and the cGMP-dependent kinase inhibit TNFalpha signaling in hepatocytes. As part of this objective, we will identify the substrates for G-kinase that mediate the protective actions. AIM II: To determine the pathways leading to efficient S-nitrosylation of caspase in hepatocytes. Under Aim II, we will identify the factors that lead to efficient S-nitrosylation of caspases in hepatocytes. Factors that are likely to be important and that will be tested include the levels of glutathione and intracellular iron as well as the ratio of NO to O2-. AIM III: To identify iNOS-induced protective genes in hepatocytes. Under Aim III, we will complete our analysis of NO-regulated genes in hepatocytes using differential display. We have already identified 11 candidate genes, and we will characterize genes that contribute to the hepatoprotective actions of iNOS. Upon completion of the three aims, it is expected that we will have an understanding of the factors that render hepatocytes less susceptible to injury and dysfunction during sepsis. By defining the protective mechanisms in hepatocytes, we will gain insights into the molecular mechanisms that lead to cellular toxicity in sepsis in susceptible organs.